It is well known that most of the bodily states in mammals, including most disease states, are affected by proteins. Such proteins, either acting directly or through their enzymatic functions, contribute in major proportion to many diseases in animals and man. Classical therapeutics has generally focused on interactions with such proteins in efforts to moderate their disease causing or disease potentiating functions. Recently, however, attempts have been made to moderate the actual production of such proteins by interactions with molecules that direct their synthesis, such as intracellular RNA. By interfering with the production of proteins, it has been hoped to affect therapeutic results with maximum effect and minimal side effects. It is the general object of such therapeutic approaches to interfere with or otherwise modulate gene expression leading to undesired protein formation.
One method for inhibiting specific gene expression is the use of oligonucleotides and oligonucleotide analogs as "antisense" agents. The oligonucleotides or oligonucleotide analogs complimentary to a specific, target, messenger RNA (mRNA) sequence are used. Antisense methodology is often directed to the complementary hybridization of relatively short oligonucleotides and oligonucleotide analogs to single-stranded mRNA or single-stranded DNA such that the normal, essential functions of these intracellular nucleic acids are disrupted. Hybridization is the sequence specific hydrogen bonding of oligonucleotides or oligonucleotide analogs to Watson-Crick base pairs of RNA or single-stranded DNA. Such base pairs are said to be complementary to one another.
Prior attempts at antisense therapy have provided oligonucleotides or oligonucleotide analogs that are designed to bind in a specific fashion to a specific mRNA by hybridization (i.e., oligonucleotides that are specifically hybridizable with a target mRNA). Such oligonucleotides and oligonucleotide analogs are intended to inhibit the activity of the selected mRNA by any of a number of mechanisms, i.e., to interfere with translation reactions by which proteins coded by the mRNA are produced. The inhibition of the formation of the specific proteins that are coded for by the mRNA sequences interfered with have been hoped to lead to therapeutic benefits; however there are still problems to be solved. See generally, Cook, P. D. Anti-Cancer Drug Design 1991, 6,585; Cook, P. D. Medicinal Chemistry Strategies for Antisense Research, in Antisense Research & Applications, Crooke, et al., CRC Press, Inc.; Boca Raton, Fla., 1993; Uhlmann, et al., A. Chem. Rev. 1990, 90, 543.
Oligonucleotides and oligonucleotide analogs are now accepted as therapeutic agents holding great promise for therapeutics and diagnostics methods. But applications of oligonucleotides and oligonucleotide analogs as antisense agents for therapeutic purposes, diagnostic purposes, and research reagents often require that the oligonucleotides or oligonucleotide analogs be synthesized in large quantities, be transported across cell membranes or taken up by cells, appropriately hybridize to targeted RNA or DNA, and subsequently terminate or disrupt nucleic acid function. These critical functions depend on the initial stability of oligonucleotides and oligonucleotide analogs toward nuclease degradation.
A serious deficiency of unmodified oligonucleotides for these purposes, particularly antisense therapeutics, is the enzymatic degradation of the administered oligonucleotides by a variety of intracellular and extracellular ubiquitous nucleolytic enzymes.
A number of chemical modifications have been introduced into antisense agents (i.e., oligonucleotides and oligonucleotide analogs) to increase their therapeutic activity. Such modifications are designed to increase cell penetration of the antisense agents, to stabilize the antisense agents from nucleases and other enzymes that degrade or interfere with their structure or activity in the body, to enhance the antisense agents' binding to targeted RNA, to provide a mode of disruption (terminating event) once the antisense agents are sequence-specifically bound to targeted RNA, and to improve the antisense agents' pharmacokinetic and pharmacodynamic properties. It is unlikely that unmodified, "wild type," oligonucleotides will be useful therapeutic agents because they are rapidly degraded by nucleases.
Potential applications of these oligonucleotides and their modified derivatives as drugs have created new challenges in the large-scale synthesis of these compounds.
The solid phase synthesis of oligonucleotides is inherently wasteful in that more than one equivalent of nucleosidic phosphoramidite synthons is used presumably to drive the reaction to completion. Given the vast amounts of oligonucleotide syntheses performed for research use and for large scale manufacture pursuant to clinical trials, the waste of expensive nucleoside phosphoramidites is a significant economic and ecological problem. This problem becomes more acute if one has to synthesize the monomer through a multistep synthesis before reaching the phosphoramidite stage, as is the case where modified sugar or nucleobase containing synthons are used in oligonucleotide therapeutic agents.
Consequently, there remains a need in the art for synthetic methods which do not require the sacrifice of large amount of phosphoramidite reagent.
The large-scale synthesis of oligonucleotide phosphorothioates can be carried out by initial formation of an internucleotidic phosphite linkage employing phosphoramidite chemistry, followed by sulfurization of the phosphite to a phosphorothioate employing a Beaucage reagent. See, e.g., Beaucage et al., Tetrahedron Lett., 1981, 22, 1859; Iyer et al., J. Org. Chem., 1990, 55, 4693; Iyer et al., J. Am. Chem. Soc., 1990, 112, 1253; Beaucage et al., Tetrahedron Lett., 1992, 48, 2223; Ravikumar et al., Nucleosides Nucleotides, 1995, 14, 1219. Nucleoside beta-cyanoethyl phosphoramidite is the most widely employed monomer synthon for this purpose, providing coupling yields of up to 98.5% with as little as a 1.5-fold molar excess of the amidite.
However, problems are commonly encountered in the automated synthesis of the oligonucleotide phosphodiesters and phosphorothioates by way of the aforementioned phosphoramidite monomer approach. One such problem is the formation of a population of shorter (deletion or failure) sequences, within which one or more intended nucleotides are absent. See, e.g., Temsamani et al., Nucleic Acids Res., 1995, 23, 1841; Fearson et al., Nucleic Acids Res., 1995, 23, 2754. The purification procedures which are routinely employed to remove deletion sequences are not 100% effective, and they fail to eliminate all impurity populations. Therefore, the final purified oligonucleotide phosphorothioate product may be contaminated with n-1 length deletion sequences, and to some extent n-2 and n-3 deletion sequences.
The formation of deletion sequences is believed to be due, in part, to inefficiency of coupling. One strategy to increase the coupling efficiency is the use of a blockmer coupling strategy, in which the coupled synthon contains two or more linked nucleotide units. See, e.g., Kumar et al., J. Org. Chem., 1984, 49, 4905; Marugg et al., Nucleic Acids Res., 1984, 12, 9095; Wolter et al., Nucleosides Nucleotides, 1986, 5, 65; Miura et al., Chem. Pharm. Bull., 1987, 35, 833. More recently, it has been demonstrated that the use of dimers can reduce the formation of deletion sequences in the oligonucleotide phosphorothioate product.
One method for synthesis of oligonucleotide phosphorothioate blockmers uses an excess of tris(2,4,6-tribromo-phenoxy)dichlorophosphorane. Hotoda et al., Tetrahedron Lett., 1987, 28, 1681; Hotoda et al., Nucleic Acid Res., 1989, 17, 5291; Wada et al., J. Org. Chem., 56, 1243; Wada et al., Tetrahedron, 1993, 49, 2043. However, the reported yields of tris(2,4,6-tribromophenoxy)dichlorophosphorane could not be reproduced by the present inventors.
There remains a need in the art for synthetic methods which do not require the sacrifice of large amount of phosphoramidite reagent, and in particular, methods providing dihalophosphoranes in improved yields would further improve the efficiency of production of oligonucleotides and oligonucleotide analogs.
The present invention addresses these, as well as other important ends.